This study was conducted to determine how 3D cultured trophoblasts' secreted factors impact NK-92 activation and cytotoxicity during early pregnancy. In this study, 6 week gestational age human cytotrophoblast stem cells (iCTB) were cultured in 2D, 3D matrigel, and 3D synthetic hydrogels composed of 20 kDa 4-arm poly(ethylene glycol)-maleimide (PEG-Mal) modified with a GFOGR adhesive ligand (1 mM) and crosslinked with dithiothreitol (DTT), a non-degradable crosslinker. On day six, trophoblast supernatants were collected to investigate the influence of trophoblast organoid secreted factors on activated NK cell phenotype, measured by CD107a expression and levels of IFNγ secretion. Here we demonstrate that NK-92 cells possess a dNK2-like phenotype, and that supernatants of cytotrophoblasts cultured in 2D and synthetic hydrogels, but not matrigel, reduce activated NK-92 cytokine secretion.